Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

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homeostasis in the periaxonal space (12). Sodium channels in astrocytes participate in Na+ homeostasis, providing a path for Na+ entry into the cytoplasm (13). The level of intracellular Na+ regulates the activity of various transporters, particularly Na+/K+ ATPase and the Na+/glutamate transporter (14,15). Astrocytes regulate water exchange between the brain and vascular space through expression of the water channel AQP4 in membrane domains in their end-feet around vessels (16). In astrocytes, voltage-gated calcium channels deliver Ca2+ into the cytoplasm and participate in the generation of glial Ca2+ signals. Astrocytes maintain the scant periaxonal ECM consisting of glycoproteins, such as laminin and proteoglycans. In the normal CNS, astrocytes express a wide variety of growth factors and receptors, many of which serve as trophic and survival factors for neurons. ONH astrocytes and lamina cribrosa cells express bone morphogenetic proteins (17), neurotrophins, and receptors (18,19).

Reactive Astrocytes

Adult, quiescent astrocytes become “reactive” after injury or disease and participate in formation of a glial scar, which does not support axonal survival or growth (20,21). The major hallmarks of a reactive astrocyte are an enlarged cell body and a thick network of processes with increased expression of GFAP and vimentin (22). Reactive astrocytes increase expression of various cell surface molecules that play important roles in cell–cell recognition and in cell adhesion to substrates, as well as various growth factors, cytokines, and receptors (23,24). Reactive astrocytes express many new ECM proteins such as laminin, tenascin C, and proteoglycans (25). The expression of TGF- (26) and TGF- (27), ciliary neurotrophic factor (CNTF) (28), fibroblast growth factor 2 (29), platelet-derived growth factor (PDGF) (30), and their receptors has been reported to induce the transition of quiescent astrocytes into the reactive phenotype or to modulate the function of reactive astrocytes. Participating in the different pathogenic mechanisms of Alzheimer’s disease (31–33), ALS (33), Parkinson’s disease (34), and multiple sclerosis (35), quiescent astrocytes become reactive in response to a wide variety of stimuli. A working definition of a reactive astrocyte in the glaucomatous ONH is that of a round, large cell with thick processes that expresses increased amounts of GFAP, vimentin, and HSP27, that is motile, and that is located either at the edge of the laminar plates or inside the nerve bundles (see Fig. 1B) (8).

CELL CULTURE MODELS OF MECHANOTRANSDUCTION IN ONH ASTROCYTES

The mechanical properties of the lamina cribrosa are complex, defined by contributions from astrocytes, lamina cribrosa cells, and blood vessels embedded in threedimensional matrix architecture. Within the lamina cribrosa, astrocytes and lamina cribrosa cells respond not only to external forces but to the stiffness of the matrix within which they reside. Cells continuously sample their mechanical microenvironment by exerting internally generated tensile forces on the surrounding matrix and adjust their phenotype in a celland tissue-specific manner in response to mechanical cues.

Transition from quiescent to reactive astrocyte occurs in response to various forms of stress such as proinflammatory cytokines and injury. Our laboratory focus is to study the transition in response to a mechanical stress, IOP. Previous work indicates

that IOP-related stress causes deformation of the lamina cribrosa, which can reach a physiologic or pathophysiologic level, depending on the individual ONH (36–39). In vivo, astrocytes in the lamina cribrosa of the ONH are exposed to a hydrostatic pressure (HP) gradient between the intraocular compartment and the extraocular optic nerve (retrolaminar tissue pressure) (40,41). The nature of the mechanical stresses that may affect astrocytes in situ might include localized shear at the membrane at sites of cell–cell or ECM–cell adhesion, HP, and compression (42,43). Many laboratories have used mechanical stimulation by physiologic or supraphysiologic HP to induce changes in genes encoding for a variety of proteins including ECM proteins, growth factors, adhesion proteins, cellular enzymes, and heat shock proteins (44–50).

SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN THE TRANSITION OF QUIESCENT TO REACTIVE ASTROCYTES

Protein Tyrosine Kinases

Protein tyrosine kinases (PTKs) are the primary mediators of the signaling network that transmit extracellular signals into the cell (51). PTK signaling activates several small G proteins, including Ras, Rap-1, and the cdc42-Rac-Rho family, as well as pathways regulated by mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase, (PI3K) and phospholipase C. Receptor-type PTKs are activated by ligand binding and directly transduce the extracellular information into intracellular tyrosine phosphorylation events, whereas non-receptor tyrosine kinases (nrPTK) function as signal transducers in concert with receptor-like molecules that lack tyrosine kinase activity. Members of the receptor PTK family include the epidermal growth factor receptor (EGFR) (52), the FGF receptor (FGFR) (53,54), the PDGF receptor (PDGFR) (30), neurotrophin receptors (55), and ephrin receptors (56,57), NrPTKs include Src, Fyn, TEC, TXK, JAK1-3, and FAK (58).

In mouse brain astrocytes, members of Src family tyrosine regulate cell adhesion through an FAK-dependent mechanism (57). Among many stimuli, PTKs are activated by mechanical stress (59). Inhibitors of PTKs, such as genistein, block the proliferation through an autocrine response of release of soluble factors in response to transmural compression in astrocytoma cells (60).

Liu and Neufeld (61) demonstrated that phosphorylation of EGFR is a necessary step for induction of nitric oxide synthase (iNOS) in astrocytes exposed to HP. Further studies on EGFR in astrocytes showed that phosphorylation of EGFR is an important upstream signal that triggers quiescent astrocytes to become reactive astrocytes in ONH in glaucoma (62,63).

EGFR, also referred to as human EGF receptor (HER) and c-erbB1, belongs to a family of four closely related transmembrane receptors with intrinsic tyrosine kinase activity. The EGFR can be activated by several ligands including EGF, transforming growth factor- , heparin-binding EGF-like protein, betacellulin, epiregulin, and amphiregulin. The binding of ligands to EGFR leads to homoor heterodimerization of EGFR with other ErbB molecules, followed by autoor trans-phosphorylation of multiple tyrosine residues in the cytoplasmic tail. This region of the activated EGFR serves as docking sites for SH2-domain-containing signaling proteins. EGFR can also

be transactivated by the activation of other membrane receptors, such as angiotensin II receptors, certain G protein-coupled receptors (GPCRs), beta2-adrenergic receptors, and insulin-like growth factor receptors. The transactivation of EGFR by other receptor pathways is caused by matrix metalloproteinase-mediated shedding of endogenous EGFR ligands (64–66). Ligand-independent tyrosine phosphorylation of EGFR may also occur (67).

HP rapidly causes tyrosine phosphorylation of EGFR in a time-dependent and pressure-dependent manner in ONH astrocytes (63). EGFR is tyrosine phosphorylated throughout the cell body and predominantly in the nucleus in astrocytes in response to HP (see Fig. 2A and B). Nuclear translocation of EGFR and its potential role as a transcription factor has been reported (68). The activation of EGFR dramatically changes the phenotypic characteristics of astrocytes. The gene transcription profile of astrocytes in response to activation of EGFR is distinctly different from that of the quiescent astrocyte. The EGFR pathway regulates a remarkable number of genes that have been documented to be related to reactive astrocytes and neural disorders (62). Most of these genes fall into categories of ECM organization, cell migration, or cytokines/cytokine receptors, suggesting that the EGFR pathway regulates cell migration, tissue remodeling, and cell–cell interaction of reactive astrocytes in glaucoma.

Studies on human glaucoma eyes confirmed that EGFR and phosphorylated EGFR are abundantly present in reactive astrocytes of the ONH with moderate to advanced optic nerve damage from patients with POAG. Phosphorylated EGFR is predominately identified in reactive astrocytes in damaged nerve bundles and in disorganized glial columns (see Fig. 2C and D). The EGFR pathway is involved in signaling the conversion of a quiescent astrocyte into a reactive astrocyte in response to elevated IOP in the ONH in glaucoma (63).

Prostaglandins Synthesis in ONH Astrocytes

Previous studies have shown that cultured astrocytes are capable of prostaglandin synthesis and release (69). Zhang and Neufeld (70) demonstrated that the activation of EGFR in optic nerve astrocytes leads to the induction of the immediate early gene cyclooxygenase-2 (COX2) and subsequent signaling through the synthesis of PGE (2). Furthermore, specific inhibition of the enzymatic activity of COX2, effectively blocked ONH astrocyte migration suggesting COX2 increased expression or activity (71). This study suggested that COX2, the “inducible” form of COX and a rate-limiting enzyme in PG synthesis, mediates basal migration in astrocytes. Previous studies showed high differential expression of PGD2 and PGE2 synthase mRNAs in ONH astrocytes isolated from glaucomatous eyes compared with normal (72). Glaucomatous ONH astrocytes also migrated faster than normal, using a similar migration assay as in this study, but without pressure stimulation. These previous studies suggested that downstream products of COX2-mediated arachidonic acid metabolism (e.g., PGE2) synthesis by reactive astrocytes could be important factors in astrocyte migration in primary responses to injury in the CNS and in secondary responses in chronic neurodegenerative disease as in glaucoma (see Fig. 3). SC58236 was recently reported

Fig. 2. (A) Immunocytochemistry for phosphorylated EGFR (p-EGFR) in cultured human optic nerve head (ONH) astrocytes in response to elevated hydrostatic pressure (HP). In control astrocytes labeled for p-EGFR, note the moderate labeling for p-EGFR and the absence of labeling in the nucleus. (B) Astrocytes exposed to elevated HP for 10 min labeled for p-EGFR. Note the enhanced labeling for p-EGFR and specific localization of p-EGFR in the nucleus. (C) Immunohistochemistry for p-EGFR in normal and glaucomatous ONHs. Normal ONHs have no positive cells for p-EGFR. (D) In glaucomatous ONHs, positively labeled astrocytes for p-EGFR (arrowheads) are abundantly present in disorganized areas in the lamina cribrosa region. Original magnifications, ×600 (from Liu B and Neufeld AH, Neurobiol Dis 2003;109–123, with permission).

to have neuroprotective activity in focal ischemia in rat brain presumably by inhibiting secondary damage because of COX2 (73).

G Protein-Coupled Receptors

Signaling through GPCRs mediates numerous cellular functions including mechanotransduction (74). ET-1 has been proposed as a mediator of reactive astrogliosis. Recent findings of increased ETbR in the processes of reactive astrocytes in human optic nerves with glaucoma provides further evidence that astrocytes are involved in the pathological mechanisms of neural injury providing histopathological evidence linking the glia-ET system with human POAG (75).

G protein-coupled endothelin-1 receptors ETA and ETB are abundantly expressed and widely distributed in ocular tissues, including the retina, optic nerve, and ONH astrocytes as described recently (76,77). In the CNC, ET-1 regulates astrocyte cell shape through tyrosine phosphorylation of FAK and paxillin (78). The effects of activation of G protein-coupled metabotropic glutamate receptors, GABA receptors, nucleotides, and catecholamines in brain astrocytes are under intense study.

Fig. 3. Effect of inhibition of cyclooxygenase-2 (COX2) in astrocyte migration. SC58236 blocked basal migration under control pressure (CP) at all times (day 1, #p < 0.05; days 3 and 5, #p < 0.005). In astrocytes exposed to hydrostatic pressure (HP) and SC58236, closure of the cell-free area (CFA) was similar as in astrocytes exposed to CP without the drug. Data represent the mean distance (in mm) ± SD migrated by drug-treated and druguntreated astrocytes exposed to CP and HP of three independent experiments. (B) Very low, if any, immunoreactivity for COX2 in the intact monolayer before denudation. (C) COX2 reactivity is detected in the cytoplasm 30 min after creation of the CFA in cells at the leading edge. (D) Intense COX2 immunoreactivity in astrocytes at the edge of the CFA and throughout the monolayer 24 h after creation of the CFA. Scale bar = 20 μm in B (refers to B–D). (from Salvador-Silva M, Aoi S, Parker A, et al. Glia 2004;45:364–377, with permission).

Ras Superfamily of Small GPCRs

The Ras superfamily members participate in many cellular processes including cell movement and act as signal transducers and/or regulators of membrane traffic (79). Ras proteins cycle between a GTP-bound state and a GDP-bound state because of their GTP hydrolysis activity and a higher affinity for GTP than GDP. In vivo, this cycle is regulated by GTPase activator proteins, which increase the rate of GTP hydrolysis, and guanine nucleotide exchange factors (GEFs), which stimulate the exchange of GDP for GTP. Members of the Rho, Rab, and Ran families are also regulated by GDP dissociation inhibitors, which, by binding to the GDP-bound form, inhibit nucleotide exchange. Rac1 and cdc42 regulate migration in astrocytes because of their ability to regulate actin cytoskeletal dynamics by signaling through effectors of the Ras-activated kinases (80–84).